PROJECT SUMMARY Understanding the functions and targets of viral noncoding RNAs (ncRNAs) may provide valuable insight into host antiviral defenses and seed new therapies. However, determining the function of longer ncRNAs is still an emerging discipline with no clear formula for success. There is an urgent need to fill in this gap in knowledge because doing so opens up new avenues for understanding virus infection and RNA biology, and may yield information on effective treatment options for virus-associated disease. We have discovered a family of long non-protein-coding RNAs (lncRNAs) shared amongst three diverse human respiratory viral pathogen families, of which the Adenovirus (AdV) virus-associated RNAs (VA RNAs) serve as the founding and prototypic members. Our long-term goal is to define the mechanisms by which ncRNA is associated with infectious and immunological disease. Consistent with this goal, our overall objective in this R21 proposal is to determine the function of a family of viral lncRNAs shared amongst different pathogenic members of the Adeno-, Polyoma, and Parvo- virus families. Our central hypothesis is that members of the family of VA-like (VA-L) ncRNAs each share structural features to inactivate common host targets including PKR and the related kinase GCN2, thereby blocking host defenses and promoting virus infection. Our hypothesis is supported by strong preliminary data demonstrating that engineering chimeric viruses to express any of the VA-L RNAs rescues defects in replication associated with mutant viruses lacking VA-L RNA expression. The rationale for this proposed research is that, once it is known how VA-L RNAs act to promote virus infection, new insights relevant to respiratory viral disease and treatments will be gained, while providing a template for future studies aimed at identifying the function of the increasing number of known viral ncRNAs. We plan to test our central hypothesis and complete the objectives outlined in this proposal via the following two specific aims: 1. Determine the relative activities and essential features of the VA-L RNAs in promoting virus infection. 2. Determine VA-L RNA host targets and their mechanism of inactivation. We expect the outcomes of these proposed experiments will provide a blueprint for future work on identifying targets of viral ncRNAs. Additionally, we expect the results from this proposed work to have an immediate impact because they will likely identify GCN2 as a previously unappreciated antiviral factor triggered by amino acid depletion that is associated with virus infection, making manipulation of GCN2 a candidate for increased production of viral vectors and/or new classes of pan-viral therapeutics. This contribution will be significant because it is expected to be of potential translational importance in the prevention of virus-associated respiratory diseases while also solving a long-standing mystery in the AdV and ncRNA fields. The research proposed in this application is conceptually innovative, because it focuses on viral lncRNA function informed by comparative evolutionary analyses combined with target identification informed by cutting edge large-scale CRISPR genetic screens.